screening pearl millet cultivars by elisa for resistance to downy mildew disease
TRANSCRIPT
Plant Pathology (1996) 45, 978-983
Screening pearl millet cultivars by ELISA for resistance to downymildew disease
S. SHISHUPALA^ V. U. KUMAR", H. SHEKAR SHETTY"* andS. U M E S H - K U M A R ""Downy Mildew Research Laboratory, Department of Studies in Applied Botany, University ofMysore, Manasagangotri, Mysore-570 006 and ''Department of Microbiology, Central FoodTechnologieal Research Institute, Mysore-570 013, India
In an earlier study, we described identification of a protein from a virulent pathotype of Sclerosporagraminicola, the binding reaction of which differentiated susceptible and resistant cultivars of pearlmillet to downy mildew disease. This protein and corresponding antibody were used in an enzyme-linked immunosorbent assay (ELISA) to screen suspension cells of pearl millet cultivars for theirresistance to the downy mildew pathogen. Screening results for 31 pearl millet cultivars correlatedpositively with the established field screening method.
I N T R O D U C T I O N
Downy mildew disease caused by Sclerosporagraminicola is a major factor affecting yieldpotential of pearl millet (Pennisetum glaucutn)hybrids. Management strategies for this disease,using high yielding resistant cultivars and geneticvariants of host populations, have not alwaysbeen successful due to resistance breakdown and/or differential behaviour of cultivars to pathogenpopulations under varying climatic conditions(SafeeuUa, 1977; Shetty & Ahmad, 1981; Ball,1983). In routine screening experiments fordowny mildew resistance programmes, the hostcultivars are often grown in pathogen-infestedsoil, with infector rows of a highly susceptiblecultivar, or under greenhouse conditions usingleaf whorl inoculation of young seedlings withsporangia/zoospores. In both cases, scoring fordisease incidence is over a 30-60-day period afterplanting (Safeeulla, 1976; Williams et al, 1981;Singh & Gopinath, 1985). Data obtained fromthese screening techniques are highly variable onaccount of environmental factors (King, 1990)and/or pathogen or host variability.
In an earlier study, we had described theidentification of a 90 kDa cell surface protein ofvirulent pathotypes of 5. graminicola, that boundspecifically to suspension cells of only thesusceptible cultivar, and its involvement in host* To whom correspondence should be addressed.Accepted 19 April 1996.
recognition (Kumar et al., 1993b). The proteinshowed differential binding specificity with cellsof pearl millet cultivars that differed in downymildew disease reaction. This suggested the useof pathogen protein in screening pearl milletcultivars for downy mildew disease resistance,that would avoid the influence of environmentalfactors. Together with the corresponding anti-body, an ELISA made the screening methodmore reliable. The details of this screeningmethod are described in this paper.
MATERIALS AND METHODS
Pathogen
A virulent pathotype of S. graminicola (Patho-type 1), maintained on a highly susceptiblecultivar of pearl millet (HB 3) in the greenhouseof the Downy Mildew Research Laboratory,University of Mysore, Mysore, India was used.
Host plant materials
Cultivars of pearl millet used in the study arelisted in Tables 1 and 2.
Callus and cell suspension cultures
Seeds of each cultivar were surface sterilised withO'l% (weight/vol.) mercuric chloride, washedrepeatedly in sterile distilled water and placed onMurashige and Skoog's (MS) medium (Murashige
Screening pearl millet for resistance to downy mildew 979
Table 1 Comparison of field" and E L I S A ' ' screening of pearl millet cultivars to downymildew disease
SINo.
123456
789
101112
1314151617
Cultivar
23 BHB37042 -DMSJ104700651BK560
Soyagon843 AAmanier
P7-4PMCD 10P 1449
IP 18298IP 18295IP 18296IP 18294IP 18292
Mean downymildew infield (%)
96-0 a94-0 a75-0 b69-0 b45-0 c35-0 d
20-0 e21-Oe14-0 f
10-0 g5-0 h1-0 i
0-0 i0-0 i0-0 i0-0 i0-0 i
Grouping basedon field
screening
Highlysusceptible
Susceptible
Resistant
Highly resistant
Absorbanceat 490nm*
± SD values
0-750 ±0-050
0-540 ±0-050
0-400 ±0-065
0-300
" Conducted in downy mildew sick plot with infector rows.'' Conducted using suspension cells, pathogen protein and antibody.* Average of two independent experiments each with five replicates (suspension cellsobtained from five different fiasks); absorbance values are above the control values.
The values in the column followed by same letter(s) are not significantly different fromeach other according to Fischer's least significant difference test (P = 0-05)
& Skoog, 1962) supplemented with 3-0 mg L~' of2,4-dichlorophenoxy acetic acid (2,4-D). Theflasks were incubated in the dark at 23±2°Cfor 30 days to obtain fresh and friable calli.
For suspension cultures, fresh pieces of calli oftest materials were transferred to 100 mL ofliquid MS-medium, supplemented with 30mgL"' of 2,4-D, in 250 mL Erlenmeyer flasks.Incubation was carried out in the dark at23 ± 2°C for 20 days on a rotary shaker (speed,lOOr.p.m,) to obtain suspension cells.
Preparation of protein extract
Sporangia were obtained from diseased leaves ofHB 3 cultivar of pearl millet. The infected leaveswere collected in the evening, washed in runningtap water to remove remnants of previous cropof sporangia, blotted dry, and incubated forsporulation. Sporangia collected from theseleaves were freeze-dried and used. For proteinextraction, they were ground with silica gel ina minimum quantity of 0-01 molL"' sodiumphosphate buffer saline (PBS; 0-85% NaCl; pH
7-2) and centrifuged at 3600 g for 30 min at 4°C,The supernatant was extensively dialysed againstdistilled water and lyophilized. The extract waselectrophoresed in a preparative SDS-PAGEand the 90kDa protein used in this study wasisolated from the gels by electroelution (Kumaret al., 1993b), Protein was quantified by the dyebinding method (Bradford, 1976),
Preparation of antiserum
Antibody raised against this protein, checked forbinding specificity with the suspension culturesof susceptible and resistant cultivars of pearlmillet as described before (Kumar et al., 1993b),was used in this study. Immunoglobulins of theantiserum obtained by ammonium sulphateprecipitation were used after extensive dialysisagainst PBS at 4°C.
Enzyme linked immunosorbent assay (ELISA)
Suspension cells of all the test plants were washedrepeatedly with sterile distilled water and the cell
980 S. Shishupala et al.
Table 2 Screening of pearl millet cultivars for downy mildew disease reaction by ELISA
SINo.
123
456789
1011
121314
Cultivar
5141 BBJ 1045141 A
843 BMakaniMBH 110P 7
841 BPNBV9-21PNBM 85213P 310-17
PMCD8IP 18297IP 18293
Absorbanceat 490nm
+ SD values
0-740±0-013 a0-710±0-007b0-715±0-006b
0-553 ± 0-009 c0-517±0-012d0-517 ± 0-008 dO-513±O-011 d
0-440 ± 0 009 e0-430 ± 0-009 e0-428 ± 0-007 ef0-425 ±0-011 f
0-270 ± 0-008 g0-278 ± 0-006 g0-272 ± 0-007 g
Groupingby ELISA
Highlysusceptible
Susceptible
Resistant
Highlyresistant
Groupingby pathogenicity*
HS (64)HS (40)HS (40)
S(25)S(20)S(15)S(12)
R(10)R(8)R(6)R(5)
HR(O)HR(O)HR(O)
* Figures in parentheses indicate percentage mean disease incidence in field trials.The values in the column followed by same letter(s) are not significantly different
from each other according to Fischer's least significant difference test {P = 0-05)
pellet was collected following centrifugation(15000 g) for 20 min at 4°C. Cell pellets of eachcultivar separately suspended in distilled, sterilewater were used. The concentration of cellsuspensions of the cultivars was made uniformby adjusting their optical density (660 nm) to 0-10absorbance and used. For ELISA, all dilutionswere made in PBS and reactions carried out at37°C. Before each reaction step, the wells werewashed with PBS three times.
One hundred microlitres of each cell suspen-sion were transferred to the wells of polyvinylchloride microtitre plates and incubated, at 37°C,for 24 h to allow the cells to bind to the wells.After washing the wells with PBS, unreacted siteswere blocked with 4% bovine serum albumin.The bound cells were reacted with 100 /xL proteinextract (10mg mL"' solution) of sporangia of5. graminicola for 60 min and the cell-boundprotein was probed indirectly by reacting withthe antibody (1:10000 dilution; 100 uL) for 2 hand biotinylated goat anti-rabbit IgG conjugate(Sigma; 1:5000 dilution; 100 uL) for 2 h. Colourfor the ELISA reaction was developed byreaction with the avidin-peroxidase conjugate(Sigma; 1:500 dilution; 100 /iL) using 0-05%H2O2 and 4-chloro 1-napthol as substrates forthe enzyme (Kumar et al., 1993b). The reactionwas stopped after 30 min with the addition of2molL"' H2SO4. Intensity of colour reaction in
the wells were read by measuring the absorbanceat 495 nm using a DYNATECH ELISA reader.Each treatment was replicated five times. Con-trols consisted of cells reacted with the proteinand treated with immunoglobulins of preimmuneserum and wells containing all additions exceptthe antibody. The dilutions of protein, antibodyand anti-antibody needed for the reactions werestandardised based on the results obtained by achecker board ELISA.
Suitability of ELISA for downy mildewscreening of pearl millet cultivars was alsoevaluated by blind experiments. For this, thecultivar reaction was first categorised by ELISAand field screening was performed to comparethe results.
Field screening for downy mildew resistance
The seeds of test plants were sown in downymildew nurseries maintained at this laboratory,containing a heavy load of soil-borne oospores ofpathotype I (~300 spores per g soil). Addition-ally, sporangial inoculum was provided by theinfector row system as described by Williams etal. (1981). The test entries were sown in arandomized block design with three replicates.Normal agronomic practices such as applicationof a basal dose of 30 kg nitrogen, 30 kg P2O5 and20 kg K2O per hectare of the field before sowing
Screening pearl tnillet for resistance to downy mildew 981
and a side dressing of 30 kg nitrogen per hectare30 days after emergence were followed to raisethe crop. A distance of 30 cm between rows and4 cm between plants was maintained and theplots were furrow irrigated at 2-week intervals oras and when required. Evaluation of differentcultivars for resistance to downy mildew wascarried out by recording the disease incidence 30days after sowing and also at the dough stage (60days). Plants were rated as diseased when theyshowed any of the typical symptoms of downymildew like stunting, chlorosis and downy growthof asexual spores on the under surface of theleaves. Percentage disease incidence was ratedfrom the number of systemically infected plants.The cultivars that showed no downy mildewsymptoms were highly resistant whilst thoseshowing 1-10%, 11-25% and over 25% diseasewere rated resistant, susceptible and highly suscep-tible respectively (Wiliams et al., 1981).
Statistical analysis
The downy mildew disease incidence of all thecultivars in field and cell reactions for antigenicprotein in ELISA were compared by Fischer'sleast significant difference test.
RESULTS AND DISCUSSION
General field screening of pearl millet underartificially epiphytotic conditions, with an infec-tor row system, resulted in heavy downy mildewincidence in susceptible cultivars (Table 1).Disease incidence ranged from 0 to 96%. Allinfected plants showed downy mildew symptomslike stunting, chlorosis and downy growth ofasexual spores on the under surface ofthe leaves.The cultivars IP 18298, IP 18295, IP 18296, IP18294 and IP 18292 were rated as highly resistantsince they recorded no disease incidence. A 1-10% disease incidence in the cultivars P 1449,PMCD 10 and P 7-4 differentiated them fromsusceptible cultivars (Amanier, Soyagaon, 843 B)showing 11-25% disease incidence and highlysusceptible cultivars (BK 560, 700651, J 104,7042 DMS, HB 3, 23 B) that recorded more than25% disease incidence.
The 90 kDa protein used in this study wasearlier identified in the cell surface of the virulentpathotype of 5*. gramirticola (Kumar et al.,1993b). Its involvement in the host-recognitionphenomenon of pearl millet downy mildew host-pathogen system was suggested due to its bindingspecificity to the suspension cells of susceptiblecultivars of pearl millet. Differential binding
reactions of the pathogen protein, as indicatedby the intensity ofthe colour reaction (expressedas absorbance at 490 nm) in ELISA, showed thatcells of the susceptible cultivars reacted stronglywith the protein when compared to cells of highlyresistant cultivars (Table 1). Highest reaction ofpathogen protein in ELISA was recorded withthe highly susceptible cultivar 23 B (showing anabsorbance of 0-750) and least binding wasobserved in the highly resistant cultivar IP18292 (absorbance 0-265). Other cultivars usedin the study recorded absorbance values between0-700 and 0-400. These absorbance values couldbe extrapolated to the susceptible and resistantnature of the cultivars as determined by fieldscreening (Table 1). Thus, it was interesting tonote that groupings based on ELISA resultscorrelated well with the routine screeningexperiments under field conditions. Statisticalanalysis revealed little difference in the standarddeviation between replicates and experiments.The ELISA results differentiating disease resis-tance in cultivars, apart from confirming ourearlier observation, were also useful in the rapidscreening of pearl millet cultivars for resis-tance to S. graminicola.
Application of this method was further testedby conducting an experiment where reaction of aset of new cultivars to S. gratninicola was firstdetermined by ELISA and the results checked byroutine field screening methods. The resultscorrelated with the ELISA values deducedbased on cultivar reaction described above andalso to the field pathogenicity test (Table 2).
The controls used for the experiment showedabsorbance due to peroxidases of the suspensioncells because heat treated cells failed to give anycolour when used as a control. This interferencecould not be avoided by using avidin-alkalinephosphatase conjugate in the reaction step, prob-ably because ofthe acid phosphatases ofthe hostcells reacting with the synthetic phosphatasesubstrate. Hence control values were deductedfrom the test values which were always higher(Table 1) to overcome the interference. This wasfound essential because preliminary experimentsshowed variations in peroxidase activity amongstcultivars, stressing the importance of maintain-ing the cells of the same cultivar for controlreactions. Whilst field screening experimentsdifferentiated minor variations in susceptibility(Table 1), such a differentiation could not bemade in ELISA reactions (Table 1) mainlybecause small variations in absorbance are notgreatly dependable in a spectrophotometric assay.
982 *. Shishupala et al.
In recent years, attention has been directedtowards in-vitro screening of host cultivarsagainst pathogen metabolites using calli, sus-pension cultured cells or isolated protoplasts(Masirevee et al., 1988; Sjodin et al., 1988; Ishida& Kumashio, 1988; Kohmoto et al., 1991). In anumber of host-pathogen interactions, resist-ance expressed in intact plants is also expressedin tissue culture under proper cultural conditions(Ingram, 1969; Warren & Routely, 1970; deZoeton et al., 1982; Trigiano et al., 1984;Mauch-Mani et al., 1989). Many immunolo-gical techniques have also been described forspecific identification, quantification and local-ization of plant pathogens (Kumar et al., 1986;Mohan, 1988; McDonald et al., 1990; Shane1991; Harrison etal., 1991; Kumar e/a/., 1993a).The screening experiment described in this paperuses suspension cells of cultivars for assay. Thus,environmental factors affecting disease develop-ment (Helgeson et al., 1976) are avoided, makingthe method more objective. Specificity of thepathogen protein binding to the cultivar com-bined with serological specificity and ELISAmakes the method sensitive and accurate eventhough this procedure does not attempt toexplain the mechanism involved in diseaseresistance. This screening system also avoidsmaintenance of downy mildew sick plots sincethe test can be performed on microtitre plates inthe laboratory. A large number of cultivars canbe screened at the same time, making research ondevelopment of resistant cultivars easy.
ACKNOWLEDGEMENTS
The authors are thankful to Indian Council ofAgricultural Research and Department ofScience and Technology, Government of India,for financial assistance. V.U.K. acknowledgesthe University Grants Commission, New Delhifor the Research Associateship award. We arethankful to Project Coordinator, All IndiaCoordinated Pearl Millet Improvement Project,College of Agriculture, Pune, India and Dr S.D.Singh, International Crops Research Institutefor Semi-Arid Tropics, Patancheru, India forkindly supplying the seeds of pearl milletcultivars used in the study.
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